Helical allophycocyanin nanotubes absorb far-red light in a thermophilic cyanobacterium

To compete in certain low-light environments, some cyanobacteria express a paralog of the light-harvesting phycobiliprotein, allophycocyanin (AP), that strongly absorbs far-red light (FRL). Using cryo–electron microscopy and time-resolved absorption spectroscopy, we reveal the structure-function relationship of this FRL-absorbing AP complex (FRL-AP) that is expressed during acclimation to low light and that likely associates with chlorophyll a–containing photosystem I. FRL-AP assembles as helical nanotubes rather than typical toroids due to alterations of the domain geometry within each subunit. Spectroscopic characterization suggests that FRL-AP nanotubes are somewhat inefficient antenna; however, the enhanced ability to harvest FRL when visible light is severely attenuated represents a beneficial trade-off. The results expand the known diversity of light-harvesting proteins in nature and exemplify how biological plasticity is achieved by balancing resource accessibility with efficiency.

The electronic coupling V (in cm -1 ) is calculated according to the ideal dipole approximation: The orientation of the TDM for the S1 state of PCB is not as well established in the literature as it is for some other chromophores (e.g., Chl molecules). For the closely related chromophore phycoerythrobilin (PEB), the TDM orientation was calculated using a CIS/3-21g level of theory to be oriented roughly along the CHD to the C4A atom (these refer to the atom names in the coordinate files for PCBs) (40). In another recent work in which the EET dynamics of the cyanobacterial PBS was modeled using Förster theory, the TDM was taken to be oriented along the axis of the conjugated parts of the bilins (11). We have calculated the orientation of the TDM for the α-PCB and β-PCBs in our system using a TD-DFT B3LYP-D3BJ/TZ2P level of theory (43)(44)(45) as described in Materials and Methods, which produces similar results. The calculated orientations of the S1 TDM of the α-PCB and β-PCB are shown in fig. S11. The calculated TDMs are almost parallel to the axis connecting the NA and the NB atom in the molecules, and this axis was therefore used for the FRET calculations. The center-to-center separation was determined as the axis connecting the CHA atoms of the donor and acceptor molecules.
Several values have been reported for the magnitude of the S1 TDM of bilins. In the EET modeling of a cyanobacterial PBS a value of 15 D was used for AP chromophores (11). Quantum mechanical calculations of the S1 TDM magnitudes of the bilins in the PE545 complex, which includes PEB and 15,16-dihydrobiliverdin, yielded magnitudes in the range of ~10-13.8 D in two different studies (40,46). In another study, quantum mechanical calculations were performed to retrieve the S1 TDM magnitudes of the PCB pigments found in C-phycocyanin and magnitudes in the range of 11.1-13.5 D were found (47). Spectral modeling of the C-phycocyanin complex yielded transition dipole moments for the pigments in the range of 5.4-6.5 D. Overall, we therefore assume that the TDM magnitude for the PCBs in FRL-AP are within the range 5. 4-15 D (47). We have therefore chosen to use a TDM magnitude that yields results that are closest to the results obtained with the pump-probe experiment, which is 10 D.
Finally, the spectral overlap of the α-PCB and β-PCB is required to calculate FRET rates, for which the emission and absorption spectrum of both are required. For the α-PCB emission spectrum, we used the steady-state emission spectrum of the FRL-AP. This is appropriate because the pump-probe experiment showed that excited-state decay arises only from α-subunits, and because the site-energies of the α-and β-PCBs are at least 1,800 cm -1 apart (~9 kBT at room temperature). Thus, the steady-state emission spectrum of the FRL-AP contains contributions from only the α-PCB. To obtain the steady-state emission spectrum of the β-PCB, we subtracted the α-PCB emission spectrum (recombinantly expressed and isolated from Synechococcus 7002) from the emission spectrum of FRL-AP containing a fraction of free β-subunits (recombinantly expressed and isolated from E. coli). The steady-state emission spectrum of the α-and β-subunits are shown in fig. S12A.
To obtain the absorption spectra of the α and β PCB chromophores, we have fitted to FRL-AP the absorption spectrum with the reversed emission spectra of the α-PCB and β-PCBs (fig. S12B), allowing the peak positions and relative amplitudes to vary. It was found that in the β-PCB absorption region the reversed disconnected β-PCB emission spectrum was not wide enough to correctly fit the data. Therefore, as an additional parameter the width of the β-PCB absorption was allowed to vary. The β-PCB emission spectrum that was used for the spectral overlap calculations was then obtained by reversing the fitted β-PCB absorption spectrum while considering a Stokes shift of 5 nm (which is the same as the determined Stokes shift of the α-PCB). The fitted absorbance spectrum is shown in fig. S12B. The area normalized spectra used for the spectral overlap calculations are shown in fig. S12C.
Using the above-mentioned parameters, formulas, and the FRL-AP structure, the Förster rates between all the pigments in the system could be calculated. Given the initial distribution of excited state populations for the pigments, the time-evolution of these populations could be calculated by solving the following master equation: Pm(t) = excited-state population of pigment m as a function of time t τd = excited-state lifetime of pigment m The excited-state lifetime τd of each pigment in the FRL-AP system was set to the value as determined by the time-resolved absorption experiment (889 ps, Fig. 6).
To simulate the EET dynamics of the time-resolved absorption experiment, the master equation was solved for the initial conditions in which the initial excitation distribution was equally divided over all the β-PCBs as described in the main text. The total simulated time evolution for the excitation densities on the α-and β-PCBs is shown in fig. S13A. Using a TDM magnitude for the PCBs of 10 D, the simulation nicely matches the experimental data. We also calculated the time in which energy is transferred along a 13-protomer nanotube. The calculated time-evolution of the excited-state populations of the α-PCBs in the system upon initial excitation of the W α-PCB is shown in fig. S13B.

Supplementary Text S2
EET efficiency in FRL-AP nanotubes.
We have evaluated the overlap integrals for EET from α-PCBs to typical Chls a and to PSI red-form Chls ( fig. S15). In order to do so, we additionally needed to determine the absorption spectra of Chl a and of the PSI red-form Chls. For the absorption spectrum of Chl a, the determined spectrum of Chl a in an Lhcb environment was taken from Cinque et al. (48) and shifted to 680 nm. The absorption spectrum for a PSI red-form Chl was modeled as a charge-transfer state and was taken from Novoderezhkin et al. (49). The peak of the spectrum was then shifted to 710 nm. Considering the proposed positioning of the FRL-AP with respect to PSI, we imagine that the energy must be extracted near the bottom of a nanotube. To estimate the upper limit of the FRL-AP antenna efficiency, we connected one of the bottom α-PCB chromophores to a perfect trap, i.e., a bottom α-PCB transfers energy with an infinite rate to the trap and there is no back-transfer. For a situation in which a single α-PCB is initially excited, we can calculate the transient population of the trap using our FRET model. The final population of the trap then represents the efficiency of EET for the given initial condition. By averaging over all α-PCB chromophores that can be individually initially excited, we find the total maximal EET efficiency of FRL-AP (fig. S15). We have considered three locations for the trap: E, G or A (the isolated α-PCB). Their calculated total maximal EET efficiencies are 40%, 46% and 32%, respectively. If we consider a FRL-AP nanotube with half the length (only PCBs N-Z are present in the system), and the trap at S, U and O, then we find EET efficiencies of 65%, 65% and 57%, respectively.                    Table S2. Cryo-EM data collection, refinement, and validation statistics for helical FRL-AP.